Thakur_2023_Biol.Reprod__

Little is known about the non-neuronal spermic cholinergic system, which may regulate sperm motility and acrosome reaction initiation process. We investigated the presence of the key acetylcholine-biosynthesizing enzyme, choline-acetyltransferase (ChAT), and the acetylcholine-degrading enzymes (AChE and BChE) and two ACh-receptors in human spermatozoa and seminal plasma. Fresh ejaculates were used for intra- and extracellular flow cytometric analysis of ChAT, AChE, BChE, alpha7-nicotinic and M1-muscarinic ACh-receptors in sperm. For deducing the source of soluble enzymes, frozen seminal samples (n = 74) were selected on two bases: 1. from vasectomized (n = 37) and non-vasectomized (n = 37) subjects, 2. based on levels of either alpha-glucosidase, fructose, or zinc to define sample subgroups with high or low fluid-contribution from epididymis, seminal vesicle respectively prostate. Flow cytometric analyses revealed that ChAT was expressed intracellularly in essentially all spermatozoa. ChAT was also present in a readily membrane-detachable form at extracellular membrane of at least 18% of the spermatozoa. These were also highly positive for intra- and extracellular BChE (>83%), and M1 (>84%) and alpha7 (>59%) ACh-receptors. Intriguingly, the sperm was negative for AChE. Analyses of seminal plasma revealed that spermatozoa and epididymides were major sources of soluble ChAT and BChE, whereas soluble AChE most likely originated from epididymides and seminal vesicles. Prostate had relatively minor contribution to the pool of the soluble enzymes in the seminal fluid. In conclusion, human spermatozoa exhibited a cholinergic phenotype and were one of the major sources of soluble ChAT and BChE in ejaculate. We also provide the first evidence for ChAT as an extracellularly membrane-anchored protein.